We studied the possible effect of flaxseed to prevent leukocytes and platelets adhesion to endothelial cells and to reduce soluble adhesion molecules (sVCAM-1) and endothelial integrity markers (vWF) in ovariectomized rats fed a high-fat diet. Forty-two female Wistar rats were either sham-operated or ovariectomized and randomly assigned for 36 weeks to three different diets: (1) low-fat diet (8% energy as fat); (2) high-fat diet (40% energy as fat, lard based, lard group); (3) high-fat diet enriched with ground flaxseed 15 g/100 g of food (40% energy as fat, lard + flaxseed group). The ovariectomized rats fed with lard + flaxseeds had significantly lower serum concentrations of sVCAM and vWF, reduced platelet adhesiveness, and lower extent of platelet and leukocyte adherence to endothelium in the histological evaluation of the aorta as compared to Ovx + lard group. In our study, high dose of ground flaxseed incorporated to lard-based diet prevented the progression of atherosclerotic lesions in estrogen deficiency rats by decreasing platelet and endothelium reactivity. Assessment of platelet adhesion, serum soluble adhesion molecule sVCAM, and endothelium integrity molecule vWF could be useful to detect the risk for atherosclerotic lesions in estrogen deficiency states and to estimate the effect of flaxseed supplementation.
In recent years, the use of foods with health protective effects, named functional foods, has received considerable attention for reducing cardiovascular disease (CVD) risk, one of these being flaxseed [
Previous studies [
The adhesion of both leukocytes and platelets to endothelial cells has been implicated in the progression of atherosclerosis and thrombus formation [
The activated endothelium plays an integral role in the development of atherosclerosis. Circulating monocytes are attracted to the endothelium by chemokines, bind to the adhesion molecules, adhere, and transmigrate to the subendothelial space, where they become macrophages, scavenge oxidized LDL, become foam cells, and contribute to the development of the fatty streak in the early stage of atherosclerosis [
The purpose of this study was to determine the possible effect of flaxseed to prevent leukocytes and platelets adhesion to endothelial cells and to reduce soluble adhesion molecules (sVCAM-1) and endothelial integrity markers (vWF) in ovariectomized rats fed a high-fat diet.
Forty-two female Wistar rats (14 weeks old, weight
All experimental procedures used in this study were in strict accordance with international ethical regulations and were approved by the Medical Ethics Committee of the Grigore T. Popa University of Medicine and Pharmacy. The experiment respected as well the instructions of the Guidelines on the Care and Use of Animals for Scientific Purposes, National Advisory committal for Laboratory Animal Research, 2004.
The animals were anaesthetized with an intraperitoneal injection of a mixture of Ketamine, doses of 100 mg/kg bodyweight and Xylazine, doses of 10 mg/kg bodyweight. Half of the rats (
The rats were kept in standard laboratory conditions, with a controlled temperature (
Diets had similar carbohydrate, total fiber, protein, and fat content (Table
Composition of experimental diets (%; w/w).
Experimental diets | 8% fat | 40% fat | 40% fat |
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Lard | Lard + flaxseed | ||
Proteins | 20.00 | 20.00 | 20.00 |
Corn starch | 62.00 | 32.00 | 32.00 |
Cellulose powder | 5.00 | 3.00 | 3.00 |
L-cysteine | 0.25 | 0.25 | 0.25 |
Vitamin mix | 1.00 | 1.00 | 1.00 |
Mineral mix | 3.50 | 3.50 | 3.50 |
Choline | 0.25 | 0.25 | 0.25 |
Fat | 8.00 | 40.00 | 40.00 |
Sunflower oil | 8.00 | 15.00 | 7.00 |
Lard | — | 25.00 | 25.00 |
Flaxseeds* | — | — | 8.00 |
After 36 weeks, the animals were sacrificed, by thiopental anesthesia (1 mL/100 g body weight from 0.01% solution), followed by opening the chest and collecting the blood by cardiac puncture. Blood samples were collected using sodium citrate as anticoagulant buffer, blood/citrate ratio of 9 : 1, or without anticoagulant. The anticoagulated blood was centrifuged (200
Serum VCAM-1 was measured by ELISA method for quantitative evaluation of human sVCAM-1 (Bender Medical System) [
Serum vWF was measured by an immunoenzymatic “sandwich" method for vWF antigen (Life Therapeutics) [
Platelet aggregation to ADP 10
Serum total cholesterol, HDL-cholesterol, and triglycerides (TG) were measured by enzymatic colorimetric methods on a TECAN microplate reader by commercially available kits (Audit Diagnostics Ireland). Non-HDL cholesterol was calculated by subtracting HDL cholesterol from total cholesterol.
For light microscopy evaluation of aortic atherosclerotic lesions, we used hematoxylin-eosin (H-E) staining.
Data were expressed as mean ± standard deviation (SD). Univariate statistical analysis was performed using the Student’s
The literature’s data supports the participation of the vascular wall in the atherogenesis process, involving an inflammatory process, endothelial dysfunction, and platelets hyperreactivity. Platelets receptors activation, platelet membrane fluidity alteration, and the membrane lipids composition change contribute to platelets activation. It has been shown that platelet activation is characterized by increased platelet adhesion and increased platelet aggregation, particularly to ADP, but also to thrombin or collagen [
In our research, the antiatherogenic mechanism of flaxseed enriched diet was investigated in ovariectomized female rats, a model of experimental atherosclerosis [
Table
Mean ± SD values for platelet functions and endothelial markers in studied groups.
Measures | Sham | Sham + lard | Sham + lard + flaxseed | Ovx | Ovx + lard | Ovx + lard + flaxseed |
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Platelet functions | ||||||
Aggregation (mOD/min) |
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Adhesion (%) |
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Endothelial markers | ||||||
sVCAM (ng/mL) |
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vWF (%) |
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Values are means ± SD,
Table
Mean ± SD values for lipid profile in studied groups.
Measures | Sham | Sham + lard | Sham + lard + flaxseed | Ovx | Ovx + lard | Ovx + lard + flaxseed |
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Serum | ||||||
Total cholesterol (mg/dL) |
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Triglycerides (mg/dL) |
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HDL-cholesterol (mg/dL) |
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Non-HDL cholesterol (mg/dL) |
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Values are means ± SD,
Examination of the aorta under a light microscope in lard-fed Ovx animals revealed signs of incipient atherosclerosis (endothelitis, leukocyte, and platelet adhesiveness and leukocyte margination, macrophage loaded with lipids in intima) (Figure
Aorta—Ovx + lard group (HE staining
Aorta—Ovx + lard + flaxseed group (HE staining
Aorta—Sham + lard + flaxseed group (HE staining
In the present study, ovariectomy and lard-based diet increased serum concentrations of total and non-HDL cholesterol, platelet aggregation and adhesion, and endothelial dysfunction markers (sVCAM-1 and vFW) and led to incipient atherosclerotic lesions in female rats fed on high-fat diet. The addition of ground flaxseed (15 g
The literature data on the hypocholesterolemic effect of flaxseeds are controversial. The experimental model of postmenopausal hypercholesterolemia and atherosclerosis, developed by Lucas et al. [
Serum HDL-cholesterol changes in flaxseed-fed sham-operated and ovariectomized animals revealed that ALA and lignans supplements had beneficial effects by slight and nonsignificant increase in HDL-cholesterol as compared to lard-fed animals. Although the differences are not significant, it confirms the beneficial effects of flaxseeds, even when two risk factors are associated. Lignans from flaxseeds may play an important role in lipid metabolism modulation; synthetic lignans significantly reduce serum cholesterol and LDL cholesterol, while increasing HDL cholesterol; it is considered that lignans modulate 7
The results of our study suggested that in a condition associated with two cardiovascular risk factors, estrogen deficiency and increased saturated fatty acids intake, the endothelial markers and platelet functions are significantly changed and the diet supplementation with flaxseed had a beneficial effect.
The lack of estrogen atheroprotection in our animal model of atherosclerosis could be connected to the state of the NO endothelial production [
High dose of flaxseed used in our study significantly reduced platelet adhesion in ovariectomized female rats. We have previously demonstrated that the addition of ground flaxseed (15 g
In our study, high dose of ground flaxseed incorporated to lard-based diet prevented the progression of atherosclerotic lesions in estrogen deficiency rats by decreasing platelet and endothelium reactivity without serum cholesterol lowering effect. Assessment of platelet adhesion, serum soluble adhesion molecule sVCAM, and endothelium integrity molecule vWF could be useful to detect the risk for atherosclerotic lesions in estrogen deficiency states and to estimate the effect of flaxseed supplementation.
Supplementing the diet with high doses of ground flaxseed may lower the atheroslerotic risk in postmenopausal women by increasing the vascular wall protection, reducing the thrombotic risk and improving the lipid metabolism.
The authors declare that there is no conflict of interests.
The research was supported by Nutrigroup Association, Iasi, Romania. The authors are grateful to Mihail Axinte and Ioan Nestian of the Phytotechny Department, Faculty of Agronomy of Iasi, for providing the flaxseeds.